1. Field of Invention
The present invention pertains to a process of obtaining protein samples having an Aurora 2 kinase catalytic domain that are amenable to forming homogenous crystals for X-ray crystallization analysis. The present invention further pertains to methods of obtaining X-ray diffractable crystals of this catalytic domain. In addition, the present invention pertains to methods of using the data from X-ray diffractable crystals of this catalytic domain in structure assisted drug design for identifying compounds that can inhibit the activity of Aurora 2 kinase or that are more drugable.
2. Background
For the year 2003, the American Cancer Society estimates the number of new cancer cases at 1,334,100 and the number of cancer related deaths at 556,500 in the United States alone. In light of the widespread number of cancer cases and cancer-related deaths, as well as the inadequacies of currently available treatments, there is a need for more effective therapeutics to treat cancer.
Cancer results from a defect in the regulation of processes that control the cell cycle. Among the proteins that control the cell cycle, members of the Aurora/Ipl1p (IPL1p) family of mitotically regulated serine-threonine kinases are emerging as key regulators. These proteins are involved in processes that ensure genetic integrity of progenic cells (centrosome maturation, chromosome segregation and cytokinesis). [Nowakowski et al., Structure (Camb), 10(12):1659-1667 (December 2002); Giet and Prigent, J Cell Sci, 112(Pt 21):3591-3601 (1999); Bischoff and Plowman, Trends Cell Biol, 9(11):454-459 (1999)].
Evidence suggests that a member of the Aurora/Ipl1p (IPL1p) family, Aurora 2 kinase (also known as Aurora-A, Aik, BTAK, STK15, ARK1 and HsAIRK1), plays a role in oncogenic transformation. [Giet and Prigent, J Cell Sci, 112(Pt 21):3591-3601 (1999); Bischoffetal., EMBO J, 17(11):3052-3065 (1998); Nigg, Nat Rev Mol Cell Biol, 2(1):21-32]. It is believed that Aurora 2 kinase mediates oncogenic transformation through centrosome amplification which thereby results in chromosomal instability. [Miyoski et al., Int J Cancer, 92(3):370-373 (2001)]. During mitosis, Aurora 2 kinase is regulated by cell cycle-dependent feedback of phosphorylation/dephosphorylation events between Aurora 2 kinase and protein phosphatase type 1 (PP1). [Katayame et al., J Biol Chem, 276(49):46219-46224 (2001)]. Deregulation of this phosphorylation/dephosphorylation pathway is believed to contribute significantly to oncogenic processes.
Evidence shows that Aurora 2 kinase is amplified and overexpressed in various human cancers, including breast, ovarian and colorectal tumors, as well as several tumor cell lines, including those of breast, ovarian, colon, prostate, neuroblastoma, and cervical. [Nowakowski et al., Structure (Camb), 10(12):1659-67 (December 2002), Cheetham et al., J Biol Chem, 277(45):42419-42422 (November 2002)].
Based on the above, Aurora 2 kinase is a promising target for use in identifying compounds to treat cancer. That is, compounds which inhibit the enzymatic activity of Aurora 2 kinase and thereby disrupt the cell cycle and proliferation of cells. [Warner et al., Mol Cancer Ther, 2(6):589-595 (2003). Structure assisted drug design is one way to optimize the success of identifying such compounds. But use of this powerful methodology requires three-dimensional structural information (e.g., as obtained via X-ray diffraction) of the target protein.
Therefore, there is a need for crystals of the Aurora 2 kinase catalytic domain that are suitable for X-ray diffraction. In particular, there is a need for crystals that are amenable to ligand exchange so as to expedite the drug design process. Along these lines, there is a need for nucleic acid constructs that encode the Aurora 2 kinase catalytic domain in a form that is amenable to formation of crystals. In addition, there is a need for purification procedures that lead to the preparation of isolated enzymatically active Aurora 2 kinase protein and/or fragments thereof. There is a need for monodisperse Aurora 2 kinase protein samples that are amenable to forming homogenous crystals for X-ray crystallization analyses. In addition, there is a need for crystals of the Aurora 2 kinase catalytic domain of sufficient quality for X-ray crystallization analyses. Furthermore, there is a need for methods for identifying inhibitors of Aurora 2 kinase through structure assisted drug design. Likewise, there is a need for methods for identifying Aurora 2 kinase compounds that are more drugable through structure assisted drug design.